Voltage-gated potassium (K) channels are critical molecular elements regulating cellular excitability and cellular signaling in nerve and muscle tissue. K channels are important in stabilizing the cell's electrical potential and terminating periods of overexcitability, such as in epileptic seizures or cardiac arrhythmias. The cloning of a variety of related K channel genes has made possible an understanding of the molecular structure and function of K channels. How does the protein transduce a voltage change into the physical opening of the pore? Which parts of the channel protein are coupled to the voltage-dependent conformation changes during activation gating? Which parts of the channel protein create or expose the open ion pore? These questions will be addressed by a combined biophysical and molecular approach using two side- specific probes of K channel gating. Biophysical studies with external zinc suggest an interaction with the molecular transitions leading to channel opening. In contrast, biophysical studies with internal 4-aminopyridine suggest an interaction with cytoplasmic regions of the open channel. Electrophysiological methods will be used to study the activity of cloned K channels expressed in frog oocytes or mammalian cells. Site-directed mutagenesis will be used to test the functional role of individual amino acids in the channel protein. Both the electrical current generated by ions flowing through open K channel pores and the current generated by the voltage-dependent molecular transitions of the channel that precede the opening of the pore will be studied. We will examine the interactions of zinc and 4-aminopyridine with the ion channel in order to learn about: (1) the mechanisms of action of two regulators of K channel function; (2) the identity of extracellular regions that influence the function of the voltage sensor; (3) the identity of cytoplasmic regions that comprise or are linked to the activation gate; and (4) the topology of voltage-gated K channels. The long-term goal of these experiments is to enrich our understanding of the molecular mechanisms by which voltage-gated K channels function. This will be useful in understanding the molecular basis for excitation and signaling in nerve and muscle and may also have therapeutic use in the design of drugs that target certain functional regions of the channel protein.